Genome engineering: new codes, new AAs, multi-virus resistance
2-3:30pm, CLSB 521 7-Nov-2012 SB204
Thanks to:
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NHGRI
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Read
LSRF
Gen9
= = = = = = = = I/O = = = = = = = Write
1
A revolution in
reading &
writing DNA
A terabyte per bite.
1944
1995
2008
4
What is a minimal replicating system ?
& why should we want one?
5
Ribozyme-Catalyzed Transcription of an
Active Ribozyme
Aniela Wochner, James Attwater, Alan Coulson, Philipp
Holliger, Science April 2011. Evolution & engineering of
an RNA polymerase ribozyme capable of synthesizing
RNAs of up to 95 nt
(& synthesis of a 27 nt
hammerhead
endonuclease ribozyme).
6
What is a minimal replicating system ?
1999 Science: Nonessential
Mycoplasma genitalium proteincoding genes: 130 of 482 (+43 tRNAs)
2006 PNAS: 67 confirmed. 34 added
26: disrupted only in M. pneumoniae
37: mixed mutant pools in liquid culture
34: Limited sampling
7
Essential
genes of a
minimal
bacterium
PNAS
2006
8
A Whole-Cell Computational Model
Predicts Phenotype from Genotype
Karr
et al.
Cell
2012
9
A Whole-Cell Computational Model
Predicts Phenotype from Genotype
Karr
et al.
Cell
2012
10
Creation of a
Bacterial Cell
Controlled by a
Chemically
Synthesized Genome
Gibson, et al 2010
Science
1 bp deletion in dnaA
Not detected: Sanger
1-kb, but wrong
clone sent by Blue
Heron. At 10-kb
level, because many
of the 454 reads
happened to end or
start near error.
11
113 kbp DNA 151 genes
Smaller.
Higher speed &
accuracy requires
a few extra genes
(E.coli 20 min.
doubling)
Reconstituted
ribosomes:
Jewett & Church
Pure translation:
Forster & Church
MSB ’05
GenomeRes.’06
Shimizu, Ueda ’01
12
Translation
23+5S : 50S
16S : 30S
E PA
13
Safe Industrial Organisms
Genome Engineering Rationale
1.
2.
3.
4.
5.
Robust, rapid doubling 10-20 minutes
Non-standard amino acids
Dependence on NSAAs
DNA non-exchangeable with environment
Multi-virus resistance
Testing essentiality of codons (13/64)
Codon
Removed
TAG
AGA/AGG
CTT/CTC
CCC
ACC
ATA
GTC
GCC
TCC
CGG
TGA
# in 47 / 290
Natural Role # in Genome Essentials
Stop
322
0
# Left
0
Arg
Leu
Pro
Thr
Ile
Val
Ala
Ser
Arg
Stop
4,228
30,030
7,401
31,766
5,797
20,757
34,747
11,672
7273
1,232
5
70
6
151
2
67
82
88
6
4
0
1*
0
0
0
0
0
0
0
0
Total
155,225
481
0 (Jul’12)
Green= 13 codon elimination
Blue = 62 codon shuffle

Lajoie,
Kosuri,
Mosberg,
Gregg,
D.Zhang
One exception of 481 codons
ACT CTT GCC
CTWG
Vsr site
highly specific
mismatch repair
Lajoie, Kosuri,
Mosberg,
Gregg, D.Zhang
4 Mbp genome CAD
Divide & Conquer Genome Engineering
19
Complete Chemical
Synthesis, Assembly,
and Cloning of a
Mycoplasma
genitalium
Genome -- Gibson,
et al 2008 Science
20
Genome Engineering:
full genome vs partial
1. E.coli > Yeast > Mycoplasma vs one species
2. Design & synthesis error consequences
3. 1E-7 genome transplantation efficiency
4. $1M vs $1 to $1k per Mb for raw DNA
Applications of in vitro translation
•Ribosome display
•Membrane protein drug receptor studies
•Personal cancer vaccines.
•Labeling one protein not the whole cell.
•New chemistries (e.g. mirror chirality)
Commercial Systems: Roche, Ambion,
Novagen, Promega, Invitrogen, Qiagen,
Stratagene, Paragon, Amersham, NEB,
Sutro, EMerck
Tony Forster
(Vanderbilt)
22
Mirror world :
Construction of Modified Ribosomes for
Incorporation of d-Amino Acids into
Proteins. Hecht lab Biochemistry 2006
A highly flexible tRNA
acylation method for
non-natural polypeptide
Synthesis.
Suga lab Nature Methods 2006
23
A vesicle bioreactor as a step toward an artificial
cell assembly --Noireaux & Libchaber PNAS 2004
eGFP no vesicle a-hemolysin-eGFP in vesicle
24
Genetically encoded unnatural amino acids
Liu & Schultz 2010 Ann Rev Biochem.
4. Orthogonal antibiotics
(ideally inexpensive)
Azide
Strained cyclo-octyne
triazole
+
Ketone
hydrazide
hydrazone
+
e.g. PEG-pAcPhe-hGH (Ambrx, Schultz) higher serum stability
Prescher, JA & CR Bertozzi (2005) Nature Chem Biol. Chemistry in living systems
Orthogonal AA chemistry (metabolic dependence)
Phosphine
Amide
Azide
+
Ketone
hydroxylamine
Oxime
+
e.g. PEG-pAcPhe-hGH (Ambrx, Schultz) higher serum stability
Prescher, JA & CR Bertozzi (2005) Nature Chem Biol. Chemistry in living systems
10X /year since 2005 (vs 1.5X for VLSI)
Carr & Church, Nature Biotech
28
4 Next-Gen Synthesis: on chips
Minimum: $500 per 1M oligos
8K Xeotron Photo-Generated Acid
12K Combimatrix Electrolytic
120K Roche, Febit Photolabile 5'protection
244K Agilent Ink-jet standard reagents
Amplify pools with flanking universal primers
6 Paths to error correction
1.Hyb-Select: Tian et al. 2004 Nature
2. MutS: Carr & Jacobson 2004 NAR
3. MutHLS: Smith & Modrich 1997 PNAS
4. Endo/Exonuclease : Bang Nat Meth. 2008
5. Errase
6. Sequencing
29
OLS bp/error
SynBIOSIS
Kosuri et al Nature Biotech
200-mer
250
130-mer
1300
130+Errase 5940
30
2 ways to Coalesce Next-Gen DNA reading & writing
Matzas, Church, et al. (Febit,HMS) Nat. Biotech Nov 2010
"perfect" part
Write
Oligo
elute
amplify
Read
sort
&
select
Select
pick
&
place
PCR
PicoTitrePlate
multiwell plate
Roche/454
micromirror
Agilent OLS
Polonator
Data
Array synthesis
Polonator
flowcell
Rolony
Photorelease
Oligonucleotide-Design
31
Integration of genome reading
& writing
Multiplex Automated
Genome Engineering
(MAGE)
Wang
MicromirrorPolonator
Terry
32
4 DNA homology-directed strategies
pKO3
E.coli
#1: ds-Circle x Circle
2 step recA+ recombination
+ Select + counterselect
Link et al J. Bact 1997
(Open-access)
#2: ds-Linear x Circle
1 step 5’>3’exo Reda/E b/T + Select
Zhang et al Nat.Gen 1998 Yu et al. PNAS 2000
(GeneBridges license)
#3: ss-90mer x ds-Circle
#4: ss-Mb x ds-Circle conjugation
Costantino &Court PNAS’03
Wang et al., Nature '09
Isaacs et al., Science ‘11
CAGE
MAGE
33
8 Optimizations for ss allele replacement
MutS 100x
Oligo length 10x
Red-b >10,000x
Co-selection 4x
Lagging strand: 30x
G > –13 kcal/mol 30x
Phosphorothioate 3x
[oligo] = 0.05-50uM
Wang, et al. Nature 2009
Isaacs et al Science 2011
Ellis et al PNAS 2001
Co-Selection: another factor of 4
Same
replichore
Locus that restores
antibiotic resistance
Cross
replichore
-Red ssDNA Allelic Replacement
mismatch
insertion
site-specific
deletion
MAGE
Primase &
Nuclease
5 changes per 2h
4E9 genomes/day/vial
Chris
Gregg
Josh
Mosberg
Marc
Lajoie
37
22705
11802
16795
8797
30462
11512
22037
7016
18894
9620
2765
18664
12210
314
1249
7490
13399 30530
15082
9540
17791 28866
21121 4810
11569
72898
32080
34568
32265
41644
12119
5733
38167
multi-virus
resistance
20899
15272
5266
New translation
code: novel AA
Safety features: no
functional DNA
exchange
39835
29581
7401
22067
9452
24106
46116
11924
2771
19820
14174
1496
21050
35252
26270
40846
24991
20813
33875
10774
15115
14901
27567
44217
54431
36108
46524
24629
Isaacs
Charalel
Church
Sun
Wang
Carr
Jacobson
Kong
Sterling
Why genome engineering?
Multi-virus resistance
Changing 13/64 codons: ACC(T), AGA(R),
AGG(R), ATA(I), CCC(P), CGG(R), CTC(L), CTT(L),
GCC(A), GTC(V), TAG(-), TCC(S), TGA(-)
Isaacs, Lajoie,
Mosberg
Kosuri,
Wang,
Carr,
et al
39
Clinical tests of non-standard AA
“orthogonal” chemistry
hydrazide
Ketone
hydrazone
+
PEG-pAcPhe-hGH Ambrx, Cho,
Schultz et al. higher serum stability
Improving a Natural Enzyme Activity through Incorporation of
Unnatural Amino Acids -- Ugwumba et al 2010 JACS
8-11-fold improvement …
in contrast to … screening
hundreds of thousands of mutants
with natural amino acids.
41
4 Selection technologies
In vitro
Clones
GC-MS
Sensorselectors
42
4 ways for in vivo coupled
sensors-selectors
1. riboswitches
3. ds-DNA
2. tRNA-ribosome
4. mRNA binding
43
68 Sensor-Selectors (old & new ligands)
56 DNA binding proteins: ada araC arcA argPR carP cpxR crp cspA
cynR cysB cytR deoR dnaA dgsA fadR farR fhlA flhCD fnr fruR fur
galR gcvA glpR hipB iclR ilvY lacI lexA lrp malT marR melR metJ
metR modE nagC narL narP ntrC ompR oxyR pdhR phoB purR
rbsR rhaS rpoE rpoH rpoN rpoS soxS tetR torR trpR tyrR
12 Riboswitches: Adenine B12 FMN Guanine Glucosamine-6phosphate Glycine di-GMP Lysine Molybdenum PreQ1 SAM SAH
TPP theophylline 3-methylxanthine
Vatsan Raman
44
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45
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